In the past several years, much attention has been given to the development of "antisense reagents" for possible therapeutic use. This is primarily due to the discovery that the root cause of many observed pathologies is a specific genetic sequence and the protein that it encodes. Such a sequence may be an abnormal segment in the nature genome of a diseased organism, or may be introduced into an organism by some foreign infectious agent. "Antisense" (or complementary) therapy involves specifically targeting and selectively binding to this specific genetic sequence.
More specifically, in the antisense approach, synthetic oligonucleotides are constructed with specific sequences to bind to mRNAs encoded for undesired proteins. Once mRNA is bound to the antisense oligomer, translation into the corresponding protein is blocked. Thus, by controlling gene expression, antisense therapy promises a class of antiviral drugs that offer a new strategy to treat human diseases.
In recent years, research in the area of antisense has experienced a dramatic surge in the wake of several concurrent advances in cell and molecular biology. Interest in the concept of antisense control was revived following the identification of naturally occurring antisense mechanisms in the genetic processes of prokaryotes. The role of native antisense RNA transcripts in regulating DNA replication in the E. coli plasmid ColE1 was demonstrated (Itoh, et al., J. Proc. Nail Acad Sci. USA 1980, 77, 2450). Subsequent investigators have implicated antisense RNA in the control of gene transcription, mRNA translation, and phage development in a variety of bacteria, phage, and plasmid systems (Green, et al., Annu. Rev. Biochem. 55: 569-597 (1986); Simons, Antisense Nucleic Acids and Proteins, Fundamentals and Applications; Mol, et al.; Marcel Dekker, Inc., (1991). There is evidence that such mechanisms may be operative in eukaryotic systems as well (Knochbin, S.; et al., EMBO J, 8: 4107. (1989)).
Another development which spurred interest in antisense research in recent years was the elucidation of the viral origin of AIDS and other diseases, including certain types of cancer. The discovery that the activity of a few key genes could have such devastating consequences brought on the realization that the search for new therapeutics must be broadened and intensified. More importantly, the recognition that these disease mechanisms operate by exploiting normal cellular genetic processes clarified the need for development of drugs of much greater specificity.
Finally, the last decade has also witnessed explosive progress in methods of chemical synthesis and sequencing of oligonucleotides (Caruthers, Science, 230: 281 (1985); Beaucage, et al. Tetrahedron, 48: 2223 (1992)). These advances in nucleic acid chemistry have increased the availability of a broad spectrum of oligonucleotide research materials. As a consequence of these recent developments, progress in the antisense field has quickly reached a point where the wide range of potential antisense applications is evident (Zon, G. Pharm. Res., 5: 539-549 (1988); Englisch, et al.; Angew, Chem. Int. Ed. Eng., 30: 613-629 (1991); Mol, et al.; Antisense Nucleic Acids and Proteins, Fundamentals and Applications; Marcel Dekker, Inc.; (1991)).
As a first step in the development of nucleic acid based pharmaceuticals, scientists have examined the potential of natural oligonucleotides as antisense therapeutics. Recent reports have concentrated on the HIV system (Goodchild, et al., Proc. Natl. Acad Sci. U.S.A., 85: 5507-11 (1988)), showing that in the presence of oligodeoxyribonucleotides complementary to viral RNA, replication of HIV is inhibited. Even while illustrating the therapeutic potential of antisense, this work also highlights two important obstacles: delivery and stability of the antisense compounds.
An oligonucleotide is a highly polar species by virtue of the negatively charged phosphodiester groups linking each nucleotide unit. Cellular uptake of mononucleotides has been shown to be nearly non-existent (Montgomery, et al., J. Med Chem., 24: 184-189 (1981)), while on the other hand, large DNA molecules were observed to enter cells (probably by pinocytosis) (Ledous, Prog. Nucl. Acid Res. Molec. Biol., 4: 23 1-267 (1965)). It is not yet clear where oligonucleotides fall in this range of behavior. It was initially assumed that the anionic character of natural oligonucleotides would render their transfer across the hydrophobic cell membrane a difficult process. However, there is now evidence that such transfer occurs as an active process modulated by specific membrane receptors (Loke, et al., Proc. Nail Acad Sci. USA 1989, 86, 3474-3478; Yakubov, et al., Proc. Natl Acad Sci. USA 1989, 86, 6454-6458). In the HIV work, as well as in many other studies of antisense activity in oocytes and embryos, the problem of oligonucleotide delivery was circumvented by direct administration of the antisense oligonucleotides via microinjection of individual cells.
The second challenge to antisense therapy arises from the observation that the inhibitory effect of antisense oligodeoxynucleotides is quickly lost due to their rapid degradation by cellular nucleases, the native enzymes responsible for hydrolysis of nucleic acids in the cell. Both of these obstacles must be addressed if the antisense strategy is to be successfully developed as a therapeutic approach.
Approaching from the perspective of organic chemistry, researchers have investigated a variety of synthetic oligonucleotide analogs designed specifically to counter the problems of hydrolytic instability and low membrane permeability presented by natural nucleic acids. These chemical approaches range from use of nucleotide stereoisomers to modification or replacement of the natural phosphodiester linking group.
The complete replacement of the phosphodiester linkage with some other connecting functionability is the most radical approach to synthesizing oligonucleotides. The low energy conformations of a new linking group must accommodate oligonucleotide geometry in which the heterocyclic bases are in the proper orientation to maintain base-pairing with a complementary strand. As might be expected, this approach has met with widely varying results.
Earlier results in this area came from the work of Halford and Jones in 1968 towards the synthesis of polymers of a thymidine analog, linked by a 3'(O).fwdarw.5'(O) carboxymethyl functionality. These researchers were perhaps the first to point out the theoretical advantage conferred by non-ionic linkages on the ability of an oligonucleotide to penetrate a cell membrane. A carboxymethyl-linked dimer of thymidine and uridine exhibited a larger hypochromic effect than either dithymidine phosphate or polyuridylic acid. This was attributed to the fact that the carboxymethyl group has one more atom than the phosphodiester linkage and therefore greater flexibility, allowing increased interaction between the nucleobases. Random length polymers of carboxymethyl-linked thymidine also showed hypochromic behavior in solution with polyadenylic acid, but not with polyuridylic acid or random sequence DNA. The success of this work led the researchers to hypothesize about the use of such analogs in modulating mRNA function in biological systems.
Workers from the Jones laboratory also investigated this novel linkage in the 5'(O).fwdarw.3'(O) reverse orientation (Bleaney, et al.; Nucl. Acids Res., 2: 690-706 (1975)). Polydeoxynucleotide analogs containing thymine, adenine, or cytosine were shown to bind their respective complementary polynucleotides, poly(A), poly(U), and poly(I). However, testing for biological activity of these compounds was hampered by their low solubility, and hydrolytic instability at pH values above 5.0.
The Birmingham group subsequently pursued the 3'(O).fwdarw.5'(C) acetamidate-linked analogs in an attempt to improve the solubility and stability of the oligonucleotides (Gait, J. Chem. Soc., Perkin I, 1389-1394 (1979)). Unfortunately, investigation of the interaction of poly(dC) and poly(T) acetamidate-linked analogs with poly(I) and poly(A), respectively, revealed no evidence of hybridization or base-stacking, in contrast with the carboxymethyl-linked polymers. The reduced flexibility of the amide group compared to the ester group apparently destabilizes the duplex-forming geometry for the acetamidate polymer. Interestingly, this conformational restriction was not evident in a model of an acetamidate-linked thymidine dimer, but became apparent in a model containing three such linkages. The 3'(O).fwdarw.5'(N) carbamate linkage was also introduced by the Jones group in 1974 (Gait, et al.; J. Chem. Soc., Perkin I, 1684-1986 (1974)). Dimers, and later, trimers prepared by Mungall and Kaiser (Mungall, et al. J. Org. Chem, 42: 703-706 (1977)), were reported to have excellent stability to chemical and enzymatic hydrolysis. The hybridization properties of carbamate-linked oligonucelotide analogs were investigated in 1987 by Weith and coworkers (Coull, et al., Tetrahedron Lett., 28: 745-748 (1987)). A hexamer incorporating the carbamate linkage was prepared from thymidine by cycles of 3-O-carbonylimidazolide formation and condensation with free 5'-amino-5'-deoxythymidine. This compound showed no ability to hybridize the complementary poly(A) or poly(dA). In comparison, the deoxycytidine analog, prepared by block synthesis of dimeric 5'-amino-2',5'-dideoxycytidine, exhibited strong binding to both poly (dG) and poly (G) (Stirchak, et al.; J. Org. Chem., 52: 4202-4206 (1987)).
Matteuci has described the synthesis of an oligonucleotide containing the "simplest and smallest isostere" of the phosphodiester group, a formacetal linkage. (Matteuci, Tetrahedron Lett., 31: 2385-2388 (1990)). This group was incorporated into a thymidine trimer used as the 3'-terminus for solid-support-synthesis of a 15-mer of thymidine and deoxycytidine of which only the last two linkages are modified. The melting profile of this oligonucleotide analog with its RNA complement was measured. The T.sub.m (a measure of the strength of the base-pairing interaction) was slightly lower than that of the unmodified oligonucleotide, but higher than that of an analog with two methoxyethylphosphoramidate linkages at the 3'-terminus. Since the analog linkage was studied only in the terminal positions of a chimeric structure containing a majority of normal linkages, little information was revealed about the inherent ability of this group to support duplex geometry.
The Nielsen group has reported the preparation of an achiral peptide nucleic acid (PNA) backbone consisting of (2-aminoethyl)glycine units with thymine bases attached via methylenecarbonyl linkers (Nielsen, Science, 254: 1497-1500 (1991); Egholm, et al.; J. Am. Chem. Soc., 114: 1895-1897 (1992)). This structure was designed by replacement of the deoxyribose phosphate backbone in a computer model of B-DNA. The binding affinity of PNA for normal DNA was found to be so strong that a PNA thymidine decamer displaced the dT.sub.10 strand of a dA.sub.10.dT.sub.10 duplex to give a hybrid with an increased T.sub.m. The researchers attribute the surprising strength of this binding to the lack of electrostatic repulsion between the duplex strands, and the constrained flexibility of the polyamide backbone. Mixed sequence PNA oligomers are currently under investigation.
To date, most of the replacements of the phosphodiester linkage that lead to an increased resistance towards nucleases are also connected with a decrease in the affinity for a complementary RNA strand. (Cohen, et al., Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc. 1989). Therefore, further investigation is needed to study replacement backbones which are both able to strongly bind to a complementary sequence of DNA or RNA, and to offer increased resistance toward nucleases. Replacement of the natural phosphodiester linkage by an amide linkage was undertaken in an effort to overcome these problems.
A study of amide based linkages replacing the phosphodiester based backbones was described in Alain De Mesmaeker, et al., "Novel Backbone Replacements for Oligonucleotides", Carbohydrate Modifications in Antisense Research, Sanghvi & Cook, Eds. 1994. Thymidine dimers having the following formulas were synthesized:
______________________________________ ##STR2## W X Y Z ______________________________________ 1 NH CO CH.sub.2 CH.sub.2 2 CH.sub.2 CH.sub.2 NH CO 3 CH.sub.2 CO NH CH.sub.2 4 CH.sub.2 NH CO CH.sub.2 5 CO NH CH.sub.2 CH.sub.2 ______________________________________
These dimers with amide based linkages were incorporated into oligonucleotides where the other linkages between the nucleosides were normal phosphodiester bonds. The affinity of the oligonucleotide to its complementary RNA strand was studied. Affinity of the corresponding oligonucleotide for an RNA target was increased. Further, the oligonucleotide's resistance towards nucleases was also increased. However, it was determined that desired thermal stability would be improved by adjusting the distance between the sugars, as well as by having a more rigid backbone.
In Jacques Lebreton, et al., "Synthesis of Thymidine Dimer Derivatives Containing an Amide Linkage and their Incorporation into Oligodeoxyribonucleotides," Tetrahedron Letters, 34 (40): 6383-6386 (1993), the synthesis of thymidine dimers with an amide group backbone is disclosed. The amide backbone is of the structure 3'-NR--CO--CH.sub.2 -5'(R.dbd.H, Me, N-Pr). On binding with complementary RNA, destabilization occurred.
In Alain De Mesmaeker, et al., "Amides as a New Type of Backbone Modification in Oligonucleotides," Angew. Chem. Int. Ed. Engl., 33 (2): 226-229 (1994), an amide function backbone in thymidine dimers of the type CH.sub.2 --NH--CO--CH.sub.2 (and isomers thereof) was disclosed as shown below: ##STR3## Increased affinity to the complementary DNA strand and RNA target and increased stability towards nucleases was discovered for the structure shown in (a) when a single amide modified backbone was incorporated in the middle of the oligonucleotide. Results for structures (b) and (c) were less satisfactory.
In Anette Chur, et al, "Synthesis of a Carboxamide Linked T*T Dimer and Its Incorporation in Oligonucleotides," Nucleic Acids Research, 21 (22): 5179-5183 (1993), a dimer containing a five atom carboxamide linker (3'-OCH.sub.2 CH.sub.2 NHC(O)-4') was disclosed. The incorporation of this dimer in oligonucleotide sequences showed moderately lowered T.sub.m values when hybridized with a complementary DNA relative to the unmodified DNA complex.
Although the amide based linkages investigated above showed increased affinity to complementary sequences and increased stability toward nucleases, the backbones investigated have four or five bond linkages, and, therefore, are relatively flexible. A flexible backbone has inherent disadvantages. Development of a more rigid backbone is desirable to provide greater binding strength during hybridization of the oligonucleotides to the complementary DNA or RNA sequence. Further, a more rigid backbone is likely to be less tolerant to mismatches to non-complementary DNA or RNA. In addition, four and five bond linkages are more difficult to synthesize than a shorter backbone. The present invention is directed to overcoming these difficulties.